Bottom Line:
We have approached this matter in a novel manner by synthesizing a library of drug-like small molecules based upon phosphorylcholine, the active moiety of the anti-inflammatory Acanthocheilonema viteae product, ES-62, which as an immunogenic protein is unsuitable for use as a drug.Following preliminary in vitro screening for inhibitory effects on relevant macrophage cytokine responses, a sulfone-containing phosphorylcholine analogue (11a) was selected for testing in an in vivo model of inflammation, collagen-induced arthritis (CIA).Testing revealed that 11a was as effective as ES-62 in protecting DBA/1 mice from developing CIA and mirrored its mechanism of action in downregulating the TLR/IL-1R transducer, MyD88. 11a is thus a novel prototype for anti-inflammatory drug development.

ABSTRACTIn spite of increasing evidence that parasitic worms may protect humans from developing allergic and autoimmune diseases and the continuing identification of defined helminth-derived immunomodulatory molecules, to date no new anti-inflammatory drugs have been developed from these organisms. We have approached this matter in a novel manner by synthesizing a library of drug-like small molecules based upon phosphorylcholine, the active moiety of the anti-inflammatory Acanthocheilonema viteae product, ES-62, which as an immunogenic protein is unsuitable for use as a drug. Following preliminary in vitro screening for inhibitory effects on relevant macrophage cytokine responses, a sulfone-containing phosphorylcholine analogue (11a) was selected for testing in an in vivo model of inflammation, collagen-induced arthritis (CIA). Testing revealed that 11a was as effective as ES-62 in protecting DBA/1 mice from developing CIA and mirrored its mechanism of action in downregulating the TLR/IL-1R transducer, MyD88. 11a is thus a novel prototype for anti-inflammatory drug development.

fig4: Lack of toxic effect of 11a on macrophages.BmMs inultralow binding tissue culture plates were rested in RPMI completemedium for 24 h before culturing with fresh medium or medium containing 11a (5 μg/mL) or ES-62 (2 μg/mL). After 18 h,the macrophages were stimulated with medium containing 100 ng/mL LPSor 10 ng/mL BLP or 0.01 μM CpG for an additional 24 h beforestaining with 7-AAD to assess their viability. The samples were analyzedby flow cytometry, and the data are presented as density plots withfrequency of 7-AAD positive (dead) cells indicated in the gates. Theresults shown are from a single experiment representative of two independentexperiments.

Mentions:
Thus, as both IL-6 and IL-12p40 (via IL-23) promote the differentiationand maintenance of Th17 responses, it was considered that 11a was most likely to mimic the protective effects of ES-62 in CIAby suppressing production of IL-17,9 acytokine that is pathogenic in RA and an emerging therapeutic target(for example via the humanized monoclonal antibody LY2439821 and thehuman monoclonal antibody AIN457; reviews refs (42,50−52)). Limited analysis ofpotency revealed that 11a was still able to induce astatistically significant reduction of LPS-stimulated production ofIL-6 by macrophages when the concentration was reduced to 1 μg/mL,but significant effects were not consistently observed at 0.2 μg/mL(data not shown). Like ES-62, 11a showed no evidenceof toxicity under conditions mimicking the macrophage screen of cytokineproduction as determined using the cell viability indicator, 7-actinomycinD (7-AAD) (Figure 4). Indeed, the SMA showedsome evidence of protecting against the loss of cell viability associatedwith exposure to LPS. 11a was thus considered suitablefor testing in vivo.

fig4: Lack of toxic effect of 11a on macrophages.BmMs inultralow binding tissue culture plates were rested in RPMI completemedium for 24 h before culturing with fresh medium or medium containing 11a (5 μg/mL) or ES-62 (2 μg/mL). After 18 h,the macrophages were stimulated with medium containing 100 ng/mL LPSor 10 ng/mL BLP or 0.01 μM CpG for an additional 24 h beforestaining with 7-AAD to assess their viability. The samples were analyzedby flow cytometry, and the data are presented as density plots withfrequency of 7-AAD positive (dead) cells indicated in the gates. Theresults shown are from a single experiment representative of two independentexperiments.

Mentions:
Thus, as both IL-6 and IL-12p40 (via IL-23) promote the differentiationand maintenance of Th17 responses, it was considered that 11a was most likely to mimic the protective effects of ES-62 in CIAby suppressing production of IL-17,9 acytokine that is pathogenic in RA and an emerging therapeutic target(for example via the humanized monoclonal antibody LY2439821 and thehuman monoclonal antibody AIN457; reviews refs (42,50−52)). Limited analysis ofpotency revealed that 11a was still able to induce astatistically significant reduction of LPS-stimulated production ofIL-6 by macrophages when the concentration was reduced to 1 μg/mL,but significant effects were not consistently observed at 0.2 μg/mL(data not shown). Like ES-62, 11a showed no evidenceof toxicity under conditions mimicking the macrophage screen of cytokineproduction as determined using the cell viability indicator, 7-actinomycinD (7-AAD) (Figure 4). Indeed, the SMA showedsome evidence of protecting against the loss of cell viability associatedwith exposure to LPS. 11a was thus considered suitablefor testing in vivo.

Bottom Line:
We have approached this matter in a novel manner by synthesizing a library of drug-like small molecules based upon phosphorylcholine, the active moiety of the anti-inflammatory Acanthocheilonema viteae product, ES-62, which as an immunogenic protein is unsuitable for use as a drug.Following preliminary in vitro screening for inhibitory effects on relevant macrophage cytokine responses, a sulfone-containing phosphorylcholine analogue (11a) was selected for testing in an in vivo model of inflammation, collagen-induced arthritis (CIA).Testing revealed that 11a was as effective as ES-62 in protecting DBA/1 mice from developing CIA and mirrored its mechanism of action in downregulating the TLR/IL-1R transducer, MyD88. 11a is thus a novel prototype for anti-inflammatory drug development.

ABSTRACTIn spite of increasing evidence that parasitic worms may protect humans from developing allergic and autoimmune diseases and the continuing identification of defined helminth-derived immunomodulatory molecules, to date no new anti-inflammatory drugs have been developed from these organisms. We have approached this matter in a novel manner by synthesizing a library of drug-like small molecules based upon phosphorylcholine, the active moiety of the anti-inflammatory Acanthocheilonema viteae product, ES-62, which as an immunogenic protein is unsuitable for use as a drug. Following preliminary in vitro screening for inhibitory effects on relevant macrophage cytokine responses, a sulfone-containing phosphorylcholine analogue (11a) was selected for testing in an in vivo model of inflammation, collagen-induced arthritis (CIA). Testing revealed that 11a was as effective as ES-62 in protecting DBA/1 mice from developing CIA and mirrored its mechanism of action in downregulating the TLR/IL-1R transducer, MyD88. 11a is thus a novel prototype for anti-inflammatory drug development.